Bone marrow stromal cells modulate osteoclast formation through direct contact or via the release of soluble cytokines such as macrophage colony-stimulating factor (CSF-1 or M-CSF). CSF-1 is a key regulatory molecule essential for proliferation of osteoclast progenitors. The importance of CSF-1 in osteoclast formation has been emphasized by its curative effect on op/op mice, a mutant deficient in CSF-1. We will determine the effect of murine TC-1 bone marrow-derived stromal cells that produce CSF-1 on osteoclast formation in Vitro. CSF-1 expression in TC-1 cells is induced by platelet-derived growth factor (PDGF) and 12- 0-tetradecanoylphorbol-13-acetate (TPA). Our focus in this proposal will be to define the molecular mechanism involved in CSF-1 gene regulation in response to PDGF, TPA and cAMP using TC-1 stromal cells as a model.
The specific aims are: 1) to determine the effect of TC-1 and other bone marrow-derived stromal cells on osteoclast formation; 2) to determine, in TC-1 cells, the effects of PDGF, TPA and cAMP on CSF-1 gene expression at the level of abundance, synthesis and stability of CSF-1 mRNA using nuclear transcript elongation assay and analysis of CSF-1 mRNA disappearance; 3) to identify and characterize cis-acting DNA elements in the CSF-1 gene that mediate its transcriptional regulation. These studies will be accomplished by transfecting TC-1 cells with chimeric plasmids which contain variably deleted 5'-flank region of the CSF-1 gene inserted upstream of a reporter gene. We will also identify trans-acting factors responsible for PDGF, TPA, and cAMP-mediated CSF-1 gene expression; 4) to determine if delivery of CSF-1 to the bone of op/op mice via autologous stromal cells that are genetically engineered to produce CSF-1, cures the osteopetrotic defect. The proposed experiments offer the potential for identifying novel regulatory mechanisms that control cytokine-induced expression of CSF-1. These studies also have the potential to expand our understanding of the role of stromal cells in regulating osteoclast formation during physiologic events, as well as pathological disease stated. Increased understanding of the molecular mechanisms by which cytokines, in general, regulate CSF-1 may lead to more effective treatment regimens designed to either enhance or inhibit the proliferation and functional activity of osteoclasts. The strategy of targeting cytokines to the bone via stromal cells represents a novel therapeutic approach with potential application to a variety of conditions such as osteoporosis and bone fracture.
